Introduction: We aimed to elucidate the inflammatory response of Aspergillus fumigatus conidia in a whole-blood model of innate immune activation and to compare it with the well-characterized inflammatory reaction to Escherichia coli. Methods: Employing a human lepirudin whole-blood model, we analyzed complement and leukocyte activation by measuring the sC5b-9 complex and assessing CD11b expression. A 27-multiplex system was used for quantification of cytokines. Selective cell removal from whole blood and inhibition of C3, C5, and CD14 were also applied. Results: Our findings demonstrated a marked elevation in sC5b-9 and CD11b post-A. fumigatus incubation. Thirteen cytokines (TNF, IL-1β, IL-1ra, IL-4, IL-6, IL-8, IL-17, IFNγ, MCP-1, MIP-1α, MIP-1β, FGF-basic, and G-CSF) showed increased levels. A generally lower level of cytokine release and CD11b expression was observed with A. fumigatus conidia than with E. coli. Notably, monocytes were instrumental in releasing all cytokines except MCP-1. IL-1ra was found to be both monocyte and granulocyte-dependent. Pre-inhibiting with C3 and CD14 inhibitors resulted in decreased release patterns for six cytokines (TNF, IL-1β, IL-6, IL-8, MIP-1α, and MIP-1β), with minimal effects by C5-inhibition. Conclusion:A. fumigatus conidia induced complement activation comparable to E. coli, whereas CD11b expression and cytokine release were lower, underscoring distinct inflammatory responses between these pathogens. Complement C3 inhibition attenuated cytokine release indicating a C3-level role of complement in A. fumigatus immunity.

A. fumigatus is one of the most widespread fungal species in nature and conidia have been found to withstand a wide range of environmental factors, such as extreme temperatures [1‒3] and a wide range of pHs [3, 4]. The fungus has a hydrophobic cell wall, which improves its dispersal abilities compared to many other fungal species [5, 6]. Therefore, A. fumigatus is easily distributed into the environment and infiltrates the human body daily. The lung is often the primary site of infection [1, 2]. Because of these characteristics, A. fumigatus is one of the most common fungal infections in humans [3, 7].

Once A. fumigatus has infiltrated the lungs, it can usually be cleared quickly by the mucosa, epithelial cells, and local alveolar immune cells [2, 8, 9]. However, immunocompromised patients have difficulties clearing A. fumigatus, which can lead to severe pulmonary infections [10]. Every year, hundreds of thousands of pulmonary infections caused by A. fumigatus are identified worldwide, particularly in immunocompromised individuals [11]. Most A. fumigatus pulmonary infections remain in the local lung tissue. However, the infection may spread across the tissue barrier and into the vascular system [7, 12]. Once A. fumigatus has spread to the bloodstream, the invasive infection has a high mortality rate of over 30% [13].

Mouse models of different complement-knockouts (C3 and C5) have shown a higher susceptibility to infections than mice lacking a functioning complement system when challenged with A. fumigatus [14]. These results are further confirmed in mice with a natural C5 defect [15]. Nevertheless, detailed knowledge about the inflammatory response against A. fumigatus in the human whole blood is lacking. The lepirudin whole-blood model is based on a highly specific inhibition of thrombin to avoid coagulation, where all other molecular and cell systems are kept open and can mutually interact [16]. The model has previously been successfully used to study the response of the complement and toll-like receptor (TLR) systems to gram-negative and gram-positive bacteria [17]. We, therefore, hypothesized that this model would also elucidate the contributions of the complement system and other facets of the initial innate immune response to A. fumigatus conidia.

Whole Blood Model

The whole blood model was performed as described by Mollnes et al. [16]. Blood was collected from healthy anonymous donors in the Blood Bank at Rigshospitalet in Copenhagen, Denmark. Whole blood was taken using venipuncture and collected into 3 mL vacuette tubes (Greiner Bio-One, Kremsmünster, Austria) with 50 μg/mL lepirudin (Refludan, Pharmion, Copenhagen, Denmark) and kept at 4°C until experiments commenced.

Samples for titration of experimental activation were taken at 0-, 15-, 30-, and 45 min. A. fumigatus was used in 1 × 104, 1 × 105, 1 × 106, 1 × 107, and 1 × 108 conidia/mL concentrations. Whole blood interactions between biological systems, including complement, were blocked in the samples by adding 20 mm EDTA before being spun at 3,000 g for 15 min at 4°C. Samples were then stored until further analysis. Samples for flow analysis were taken before EDTA addition and centrifugation of samples. Heat-inactivated E. coli (ATCC-33572, Merck Life Science, AS, Oslo, Norway) was used as a positive control at 107E. coli/mL concentration.

Cultivation of Aspergillus fumigatus

In-house cultivation of A. fumigatus was possible due to a generous gift of the clinically isolated strain (6871) by Prof. Luigina Romani at the University of Perugia (Institute of Infectious Diseases). This strain was isolated from an in-patient case of invasive aspergillosis, resulting in a fatal outcome. A. fumigatus was cultivated in plates containing Sabouraud agar (32.5 g Sabouraud Glucose Agar with Chloramphenicol (Sigma-Aldrich, Saint Louis, MO) in 500 mL aqua dest.). After formulation of the agar, it was autoclaved at 121°C for about 15 min and aliquoted into petri dish plates while still liquid. After cooling to room temperature, the agar could harden and be stored at 4°C for up to 4 weeks.

The isolated and active strain was added as three equally spaced droplets of 10 μL to each dish to inoculate plates. The fungi were then incubated at 37°C for approximately 4 days, after which one colony was used to inoculate a new plate. This re-inoculation was repeated until uniform growth was achieved.

The uniform colony and its spores were then harvested by adding 50 mL of PBS with 0.01% Tween and 150 mm NaCl to the plate in 5 mL increments. Conidia were scraped off the plate with a cotton tip and solubilized with every step. To extract only spores and not hyphae or other growth stages of the fungi, the solution was filtered using a 40 μm cell strainer (BD Biosciences, BD Franklin Lakes, NJ). Finally, the solution was washed three times with PBS with 0.5% Tween and resuspended in PBS with 0.01% Tween and 150 mm NaCl. After microscopy verification to confirm that conidia were resting (7, 18), the spores were heat inactivated at 121°C for 15 min. Spores were aliquoted at 1.25 × 109 cells/mL and stored at −80°C for further use.

Cell Depletion

Depletion of individual leukocyte cell populations was done according to a method described in detail elsewhere by Fageräng et al. [18]. Briefly, lepirudin whole blood with 0.07% citrate was split into platelet-rich plasma and a blood cell pellet by spinning samples at 120 g for 15 min at 20°C with no breaks. The blood cell pellet was mixed with magnetic StraightFrom® Whole Blood Microbeads (Miltenyi Biotec Norden AB, Lund, Sweden) for CD14 (a monocyte marker) and CD15 (a granulocyte marker) analysis and separated over a column. Per 1 mL of whole blood, 50 μL of beads were added. Blood pellet and platelet-rich plasma were recombined in 1:1 and reactivated with a final concentration of 6.25 mm Ca2+ [19]. The whole blood model was then performed on samples according to the above instructions. Sample activation was stopped with 20 mm EDTA and then centrifuged at 3,000 g at 4°C for 15 min rWB-ctrl indicates control reconstituted whole blood, rWB-CD14dpl indicates whole blood lacking (dpl a.k.a. depleted) CD14+ monocytes, and rWB-CD15dpl indicates whole blood lacking CD15+ granulocytes. Double Depletion of CD14+ and CD15+ cells was done using the same protocol but the CD14 and CD15 beads were combined in the same sample, this sample is indicated as rWB-CD14dpl/rWB-CD15dpl.

Complement Inhibition

For complement inhibition of samples, 10 μm CP40 to inhibit C3 (a gift from Prof. John Lambris, Philadelphia, PA) and 100 μg/mL eculizumab to inhibit C5 from Alexion Pharmaceuticals (Zurich, Switzerland), was used respectively. Fifteen μg/mL mouse monoclonal anti-CD14 r18d11 was used for CD14 inhibition. The mouse monoclonal anti-CD14 r18d11, was produced in our laboratory and described in detail was used to inhibit the CD14-dependent toll-like receptor pathway [20]. As a control treatment, 10 μm scrambled control peptide [21] and 15 μg/mL isotype control antibody NHDL [22] were included. Lepirudin whole blood was pre-incubated with the indicated inhibitors for 5 min before adding 1 × 107 conidia/mL to the blood. Samples were then incubated for 0, 30 or 120 min before their activation was stopped by using 20 mm EDTA. Samples were spun at 3,000 g for 15 min at 4°C and plasma was stored frozen until further analysis. Flow cytometry samples were taken before EDTA addition.

Flow Cytometry

To prepare whole blood samples for flow cytometry, samples were first treated with a high-yield lyse solution (Invitrogen, Waltham, MA) to achieve lysis of erythrocytes. Samples were then incubated with the antibodies of choice for 15 min at 4°C in the dark. For whole blood analysis, the following antibodies were used: CD45 and CD14 were studied using a BD Simultest containing anti-CD45 mAbs FITC and anti-CD14 mAbs PE conjugated (BD life science), anti-CD15 (clone H198) conjugated with eFlour 450 (Invitrogen), and an anti-CD11b (clone ICRF44) conjugated with PE-Cy 5 (BioLegend, San Diego, CA). Samples were fixed with 0.01% paraformaldehyde (v/v). Results were analyzed using BD FACS DIVA (BD life science) software.

A gate of 10,000 CD45+ cells was selected using SSC-A/CD45+. Single cells were gated using SSC-A/SSC-H. CD14+ cells were gated using SSC-A/CD14+ and CD15+ cells using SSC-A/CD15+ of the SSC-A/CD45+ positive population. CD11b was measured as MFI of individual populations found on the SSC-A/CD14+ and SSC-A/CD15+ gates. Results were analyzed using Kaluza analysis software (Beckman Coulter, Brea, CA). CD14+ and CD15+ population cell counts were defined as number of cells with indicated marker per 10,000 CD45+ cells. For the comparison of depletion samples, a stopping gate was placed on the CD14 and CD15 double negative population to retain a reliable comparison between the sample types.

Analysis of Soluble Markers

Complement activation was analyzed by measuring sC5b-9 as described previously [23] and later modified in [24]. Briefly, Monoclonal anti-human C9 clone aE11 (produced in-house) [25] was used to capture sC5b-9, and monoclonal anti-human C6 clone 9C4 (produced in-house) was used for detection. Samples were diluted 1:5 in AG buffer (PBS with 0.2% Tween20 and 0.02 M EDTA) and were read at 450 nm using a Synergy HT microplate reader (BioTek, Winooski, VT).

For analysis of cytokines, plasma samples from the whole blood model of inflammation were thawed on “slush-ice.” The samples were then subjected to the Bio-plex Human Cytokine 27-Plex multiplex assay (Bio-Rad Laboratories, Hercules, CA, USA), which includes: IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, bFGF-2, Eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF, RANTES, TNF (formally known as TNFα), and VEGF, according to manufacturer’s instructions. Samples were analyzed using the Luminex 200 platform (R&D Systems, MN).

Statistics

Results were analyzed using GraphPad Prism 9.5. Samples comparing the titration of A. fumigatus over time were analyzed using Friedman’s test. The rest of the results were analyzed using Kruskal-Wallis test. Graphs show median ± interquartile range. Significant results were defined as p < 0.05. All experiments were done in at least n = 6.

Complement Activation Detected by sC5b-9 Formation following A. fumigatus Incubation in Whole Blood

Titration of 107 conidia/mL A. fumigatus at 0-, 15-, 30- and 45-min post-incubation showed a significant increase in sC5b-9 formation at 30-min post-stimulation (p = 0.047) and further slightly increased after 45 min (Fig. 1a). This increase was similar to incubation with 107E. coli/mL and no significant difference in activation was found between the two microbes at any time point (p > 0.05). The sC5b-9 formation was also studied at 30 min post-incubation with concentrations of 104, 105, 106, 107 and 108 conidia/mL (online suppl. Fig. S1A; for all online suppl. material, see https://doi.org/10.1159/000539368). A significant increase in sC5b-9 formation after A. fumigatus incubation as compared to 107E.coli/mL was found following incubation with 107 (p = 0.011) and 108 conidia/mL (p < 0.0001).

Fig. 1.

Complement and leukocyte activation in whole blood following A. fumigatus and E. coli incubation. Complement activation product sC5b-9 (a), monocyte CD11b (b) and granulocyte CD11b (c) expression are shown over time of incubation. Stars above the bar indicate significant change compared to whole blood time zero (0) control using the Friedman test. Bars show median ± interquartile range. Single dots represent individual donors. Significance indicated on graphs as: * for p < 0.05, ** for p < 0.01, for p < 0.001, and **** for p < 0.0001.

Fig. 1.

Complement and leukocyte activation in whole blood following A. fumigatus and E. coli incubation. Complement activation product sC5b-9 (a), monocyte CD11b (b) and granulocyte CD11b (c) expression are shown over time of incubation. Stars above the bar indicate significant change compared to whole blood time zero (0) control using the Friedman test. Bars show median ± interquartile range. Single dots represent individual donors. Significance indicated on graphs as: * for p < 0.05, ** for p < 0.01, for p < 0.001, and **** for p < 0.0001.

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CD11b Expression following A. fumigatus Incubation in Whole Blood

CD11b expression after A. fumigatus stimulation was analyzed on the CD14+ (monocytes, Fig. 1b) and CD15+ (granulocytes, Fig. 1c) cell populations using flow cytometry. Titration of 107 conidia/mL A. fumigatus at 15-, 30-, and 45-min post-stimulation showed a significant increase in CD11b expression at 30-min post-incubation (p = 0.013) on CD14+ cells. Titration of 107 conidia/mL A. fumigatus at 15-, 30-, and 45-min post-incubation on CD15+ cells showed a significant increase in CD11b expression at both 30- (p = 0.027) and 45-min (p = 0.027) post-incubation.

Titration with concentrations of 104, 105, 106, 107 and 108 conidia/mL A. fumigatus incubated for 30 min only showed significant increase in CD11b expression on CD14+ cells for 107 conidia/mL (online suppl. Fig. S1B). Titrations with the same concentration on CD15+ cells showed a significant increase in expression with both 107 (p = 0.018) and 108 (p = 0.0009) conidia/mL (online suppl. Fig. S1C).

The count of CD14+ and CD15+ cells per 10,000 CD45+ cells showed a substantial drop in CD14+ cells at 108 conidia/mL (online suppl. Fig. S1D), making analysis of CD11b expression on CD14+ cells difficult at this concentration.

Cytokine Expression following A. fumigatus Incubation

Following whole blood incubation with 107 conidia/mL A. fumigatus for 2 h, 13 cytokines in a 27-multiplex kit were elevated statistically significant (Fig. 2, 3). TNF (Fig. 2a), IL-1β (Fig. 2b), IL-4 (Fig. 2c), IL-6 (Fig. 2d), IL-17 (Fig. 2e), IFN γ (Fig. 2f), IL-1ra (Fig. 2g), IL-8 (Fig. 3a), MCP-1 (Fig. 3b), MIP-1α (Fig. 3c), MIP-1β (Fig. 3d), FGF-basic (Fig. 3e), and G-CSF (Fig. 3f) were elevated in A. fumigatus incubated whole blood compared to the PBS control. Positive control of 107E. coli/mL was included for each. The results showed a generally lower level of cytokine release with A. fumigatus incubation than the positive E. coli control.

Fig. 2.

Release of classical cytokines following PBS, A. fumigatus, and E. coli incubation. The release of the cytokines TNF (a), IL-1β (b), IL-4 (c), IL-6 (d), IL-17 (e), IFN γ (f), and IL-1ra (g) are show as bars with median ± interquartile range. Single dots represent individual donors. Significance was calculated using Kruskal-Wallis tests. Significance indicated on graphs as: * for p < 0.05, ** for p < 0.01, ***for p < 0.001, and **** for p < 0.0001.

Fig. 2.

Release of classical cytokines following PBS, A. fumigatus, and E. coli incubation. The release of the cytokines TNF (a), IL-1β (b), IL-4 (c), IL-6 (d), IL-17 (e), IFN γ (f), and IL-1ra (g) are show as bars with median ± interquartile range. Single dots represent individual donors. Significance was calculated using Kruskal-Wallis tests. Significance indicated on graphs as: * for p < 0.05, ** for p < 0.01, ***for p < 0.001, and **** for p < 0.0001.

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Fig. 3.

Release of chemokines and growth following PBS, A. fumigatus, and E. coli incubation. The release of IL-8 (a), MCP-1 (b), MIP-1α (c), MIP-1β (d), FGF-basic (e) and G-CSF f) are shown as bars with median ± interquartile range. Single dots represent individual donors. Significance was calculated using Kruskal-Wallis tests. Significance indicated on graphs as: * for p < 0.05, ** for p < 0.01, ***for p < 0.001, and **** for p < 0.0001.

Fig. 3.

Release of chemokines and growth following PBS, A. fumigatus, and E. coli incubation. The release of IL-8 (a), MCP-1 (b), MIP-1α (c), MIP-1β (d), FGF-basic (e) and G-CSF f) are shown as bars with median ± interquartile range. Single dots represent individual donors. Significance was calculated using Kruskal-Wallis tests. Significance indicated on graphs as: * for p < 0.05, ** for p < 0.01, ***for p < 0.001, and **** for p < 0.0001.

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According to the previously categorized method [18], specific depletion of individual leukocyte populations was done to determine the cell dependency of individual cytokines (Fig. 4, 5). The efficiency of CD14 and CD15 depletion was found to be above 90% for both CD14+ monocytes (online suppl. Fig. S2A) and CD15+ granulocytes (online suppl. Fig. S2B) (as seen by flow cytometry count of CD14+ and CD15+ marker). The results do not show the definite source of the cytokine release, but does indicate the total role (including direct and indirect effects) of individual cell populations. Of the 13 cytokines that were significantly elevated following A. fumigatus conidia incubation, 11 (TNF, IFN γ, IL-1ra, IL-4, IL-6, IL-17A, IL-8, MIP-1α, MIP-1β, FGF-basic and G-CSF) appeared to be dependent only on the CD14+ monocytes for cytokine release. This observation was based on a significant change in cytokine release between rWB-ctrl and rWB-CD14dpl (p < 0.05), but not between rWB-ctrl and rWB-CD15dpl. IL1ra (Fig. 4g) showed dependency on both the CD14+ monocytes and CD15+ granulocytes, indicated by a significant change in cytokine release when comparing rWB-ctrl to both rWB-CD14dpl (p = 0.031) and rWB-CD15dpl (p = 0.006). Only 1 cytokine, MCP-1 (Fig. 5b), showed no significance on either CD14+ monocytes or CD15+ granulocytes, indicated by no difference in cytokine release between rWB-ctrl and rWB-CD14dpl (p = 0.07) or rWB-CD15dpl (p = 0.11). To confirm the results of the CD14+ and CD15+ single depletions, a dual depletion of both CD14+ and CD15+ was conducted. The % cytokine release compared to the individual controls can be seen in online supplementary Figures S3 and S4.

Fig. 4.

Role of CD14+ monocytes and CD15+ granulocytes in the release of classical cytokines following PBS, A. fumigatus, and E. coli incubation. The release of cytokines TNF (a), IL-1β (b), IL-4 (c), IL-6 d), IL-17 (e), IFN γ (f), and IL-1ra (g) are shown for reconstituted whole blood control (rWB-ctrl), reconstituted whole blood depleted for monocytes (rWB-CD14dpl) and reconstituted whole blood depleted for granulocytes (rWB-CD14dpl). Bars show median ± interquartile range. Significance was calculated using Kruskal-Wallis tests. Significance indicated on graphs as: * for p < 0.05, ** for p < 0.01, for p < 0.001, and **** for p < 0.0001.

Fig. 4.

Role of CD14+ monocytes and CD15+ granulocytes in the release of classical cytokines following PBS, A. fumigatus, and E. coli incubation. The release of cytokines TNF (a), IL-1β (b), IL-4 (c), IL-6 d), IL-17 (e), IFN γ (f), and IL-1ra (g) are shown for reconstituted whole blood control (rWB-ctrl), reconstituted whole blood depleted for monocytes (rWB-CD14dpl) and reconstituted whole blood depleted for granulocytes (rWB-CD14dpl). Bars show median ± interquartile range. Significance was calculated using Kruskal-Wallis tests. Significance indicated on graphs as: * for p < 0.05, ** for p < 0.01, for p < 0.001, and **** for p < 0.0001.

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Fig. 5.

Role of CD14+ monocytes and CD15+ granulocytes in the release of chemokines and growth factors following PBS, A. fumigatus, and E. coli incubation. The figure shows the release of molecules IL-8 (a), MCP-1 (b), MIP-1α (c), MIP-1β (d), FGF-basic (e), and G-CSF (f). Bars show median ± interquartile range. Significance was calculated using Kruskal-Wallis tests. Significance indicated on graphs as: * for p < 0.05, ** for p < 0.01, for p < 0.001, and **** for p < 0.0001.

Fig. 5.

Role of CD14+ monocytes and CD15+ granulocytes in the release of chemokines and growth factors following PBS, A. fumigatus, and E. coli incubation. The figure shows the release of molecules IL-8 (a), MCP-1 (b), MIP-1α (c), MIP-1β (d), FGF-basic (e), and G-CSF (f). Bars show median ± interquartile range. Significance was calculated using Kruskal-Wallis tests. Significance indicated on graphs as: * for p < 0.05, ** for p < 0.01, for p < 0.001, and **** for p < 0.0001.

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Complement Inhibition Effects on A. fumigatus-Induced Cytokine Release

To determine the role of complement in the cytokine response to A. fumigatus, whole blood was pre-incubated with the C3-inhibitor CP40, the C5-inhibitor eculizumab, anti-CD14 Ab r18d11, CP40 control peptide and NHDL isotype control before incubation with A. fumigatus (Fig. 6). Following treatment, 6 cytokines, IL-1β (Fig. 6a), IL-6 (Fig. 6b), IL-8 (Fig. 6c), TNF (Fig. 6d), MIP-1α (Fig. 6e) and MIP-1β (Fig. 6f) showed altered cytokine release compared to non-inhibited samples (defined as 100% and shown using stippled line on the figure). The values of the individual treatments (in pg/mL) can be found in online supplementary Table 1 along with a comparison of the results compared to the A. fumigatus stimulation alone. Following comparison of the cytokine change and the controls (Ctrl Peptide and NHDL), the combination of C3- and CD14-inhibition was found to be significantly reduced for all cytokines. Furthermore, the C3-inhibition alone was significantly reduced for IL-6, IL-8, TNF and MIP-1α. These results strongly indicate a complement effect on the C3-level rather than the C5-level. Eculizumab and CD14 co-inhibition significantly reduced IL-8 release.

Fig. 6.

Role of complement molecules in the release of cytokines following A. fumigatus incubation. Samples were treated with CP40 (C3 inhibition), eculizumab (C5 inhibition), a-CD14 (CD14 inhibition, Ab clone r18d11), and control peptide (CP40 control) and NHDL (IgG2/4 isotype control). Significant inhibition was seen for IL-1β (a), IL-6 (b), IL-8 (c), TNF (d), MIP-1α (e), and MIP-1β (f). Results are shown as percent change of release following inhibition compared to A. fumigatus stimulation without inhibition (defined as 100% and illustrated with a stippled line). Bars show median ± interquartile range. Significance was calculated using Kruskal-Wallis tests and indicates a significantly altered result compared to control. Significance indicated on graphs as: * for p < 0.05 and ** for p < 0.01.

Fig. 6.

Role of complement molecules in the release of cytokines following A. fumigatus incubation. Samples were treated with CP40 (C3 inhibition), eculizumab (C5 inhibition), a-CD14 (CD14 inhibition, Ab clone r18d11), and control peptide (CP40 control) and NHDL (IgG2/4 isotype control). Significant inhibition was seen for IL-1β (a), IL-6 (b), IL-8 (c), TNF (d), MIP-1α (e), and MIP-1β (f). Results are shown as percent change of release following inhibition compared to A. fumigatus stimulation without inhibition (defined as 100% and illustrated with a stippled line). Bars show median ± interquartile range. Significance was calculated using Kruskal-Wallis tests and indicates a significantly altered result compared to control. Significance indicated on graphs as: * for p < 0.05 and ** for p < 0.01.

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Prior studies have revealed that alveolar macrophages, epithelial cells, dendritic cells, and incoming granulocytes are pivotal in managing and eradicating A. fumigatus infection [9]. In immunocompetent individuals, the combined action of epithelium [26], alveolar macrophages [27], and neutrophils [28], among others, ensures effective clearance of fungal conidia and hyphae from the lung. Especially alveolar macrophages are instrumental in the early cytokine release within the lung in mobilizing immune cells (such as macrophages/monocytes, neutrophils, and dendritic cells) from the bloodstream to the lung as well as in inducing effector functions [29, 30].

Our study sought to understand the impact of A. fumigatus conidia on the initial inflammatory response utilizing an established human lepirudin whole blood model as proxy of blood components present in the lung [16]. Past research indicates a crucial role of the complement system in clearing A. fumigatus [15, 31‒33]. Hence, employing a model that preserves the complete complement functionality both on the soluble and cellular level would increase our understanding of the initial host response to the fungus. After titrating A. fumigatus conidia, we found that 107 conidia/mL and a 30-min incubation were sufficient to detect complement activation and CD11b expression. This concentration is in accordance to what is used in intravenous inoculation of animal models [34], indicating a comparable effect across species and a relevance to the concentration used. No discernible difference was observed between A. fumigatus and E. coli stimulation regarding complement activation or CD11b expression. This suggests that both A. fumigatus conidia E. coli have similar complement activation potential. Upon increasing the concentration of A. fumigatus past 107 conidia/mL a substantial decrease in cell count of CD14+ monocytes and a smaller change in CD15+ granulocytes was found. This is in accordance with previous research that has shown an effect of A. fumigatus virulence factors on both the monocyte and neutrophil populations at high concentrations [35, 36].

When exploring the release of cytokines following A. fumigatus conidia incubation in whole blood, a significant increase in the release of 13 cytokines was found. The cytokines released in our model matched previous studies showing cytokines released in the lung by alveolar macrophages [29, 30]. Activation of the respiratory environment through pattern recognition receptors has revealed the release of cytokines such as TNF, IL-1β, IL-6, IL-8, IL-17, MIP-1α and MCP-1 [7, 37]. All these cytokines are found to be elevated in the model used in this study, indicating preserved cytokine release across tissues. Furthermore, the similar cytokine responses found in the lepirudin whole blood further strengthens the versatility and accuracy of the lepirudin whole blood model.

However, a substantial difference in the levels of released cytokines was found when comparing similar concentrations of A. fumigatus and E. coli. This contrasts the CD11b and sC5b-9 activation results, where comparable activation levels were found when stimulated with A. fumigatus and E. coli. This is an interesting distinction between different immune effectors early in the immune response. Moreover, it further demonstrates the potentially important role complement plays in the immune response to A. fumigatus, as the activation of complement with opportunistic fungi is on par with activation to generally pathogenic bacteria. These results differentiate how distinctive immune responses are to individual pathogens and how different immune effectors play different roles in response to different microorganisms.

As an extension of the cytokine release, we examined whole blood depleted for leukocyte cell populations to identify the leukocyte cell population(s) responsible for the various cytokines released. Previous studies have determined that alveolar macrophages, neutrophils and monocytes recruited from the blood are essential in containing and removing A. fumigatus from the lung [27, 28]. Using a model designed to remove individual cell populations from the whole blood, which causes minimal complement, leukocyte, or platelet activation [18] we removed the CD14+ monocyte and CD15+ granulocyte population (Fig. 4-5). Using our new model, we could show an extensive role for monocytes in early immune response toward A. fumigatus conidia. Of the 13 cytokines significantly increased in response to A. fumigatus, 11 of these cytokines (TNF, IL-1β, IL-4, IL-6, IL-17A, IFN γ, IL-8, MIP-1α, MIP-1β, FGF-basic, and G-CSF) proved to be highly monocyte dependent. IL-1ra was shown to be both monocyte- and granulocyte-dependent, while MCP-1 was neither monocyte nor granulocyte-dependent. These results differ slightly from those previously found for E. coli, where cytokines IL-1β, IL-6, and IL-8 were also granulocyte dependent [18] and could indicate that the granulocyte role in cytokine release potentially varies in response to different activation sources [38]. These findings illustrate that the inflammatory responses to various microorganisms differ and the roles of effector functions in the innate immune response are not uniform. Neutrophils have been shown to be pivotal in the immune defense against A. fumigatus in the lungs [28, 39]. Yet, their role appears not to be linked to cytokine release as indicated in the present study. The role of neutrophils in the defense against A. fumigatus conidia could be more linked to the reactive oxygen species (ROS) system, a pathway that has previously been shown to be important for A. fumigatus clearance [37]. The cytokine release pattern of monocytes in response to A. fumigatus mirrors that seen of alveolar macrophages from earlier studies [29, 30]. This suggests that the model does accurately depict the cell populations’ contribution to cytokine release, which could further indicate an altered role of neutrophils to E. coli and A. fumigatus. The accuracy of the single depletion of CD14+ and CD15+ cells could be confirmed following the dual depletion of CD14+ and CD15+ cells as the results were very comparable to that found in the single depletions. This indicates not only an accurate representation of the function of the individual cell populations but also reiterates the strength of the depletion model itself.

Prior research has established the significance of the complement system in the immune response against A. fumigatus [15, 31‒33]. Consequently, we aimed to understand its role in cytokine release. We pre-incubated whole blood with specific inhibitors against C3 (CP40), C5 (eculizumab), and CD14 (mAb r18d11) and control treatments against the CP40 peptide (ctrl peptide) and isotype control against eculizumab and a-CD14 (NHDL). CD14 is a coreceptor for TLR2 and TLR4, and so inhibition of CD14 can provide input on the role of TLR and MYD88 signaling in the immune response to A. fumigatus [40]. The complement inhibitors, known to be effective in whole blood without unintended side effects, have been used in various studies. For instance, the C3-inhibitor CP40 has been tested in trials for diseases with excessive complement system activity [41, 42]. Eculizumab, a C5-inhibitor, was the first complement therapy approved and has been used in various diseases [43, 44]. The combined inhibition of C3 or C5 and CD14 has demonstrated strong dual inhibition of inflammation [45], but no therapeutic trials have been conducted for this combination.

When studying the cytokine release after complement inhibition, we noted significant changes in six cytokines: TNF, IL-1β, IL-6, IL-8, MIP-1α, and MIP-1β. The results found in this paper suggest a C3-mediated effect in early immune responses post-A. fumigatus infection rather than a C5 effect. This can be seen in Figure 6 as the dual C3 and CD14 inhibition significantly altered the release of all 6 cytokines compared to the control. Furthermore, IL-6, IL-8, TNF, and MIP-1α show significantly altered cytokine release following C3 inhibition alone. Dual C5 and CD14 inhibition also significantly altered IL-8 release.

Previous studies have identified TNF [46], IL-6 [47], and through indirect pathways IL-1β [48] as pivotal in the lung’s immune response to A. fumigatus. IL-8 has been shown to be strongly induced following fungal infection, but in a TLR-independent pathway [49]. The results from this study indicate that the IL-8 induction could possibly be through a complement-dependent pathway. The fact that many cytokines essential for immune responses to A. fumigatus are affected by complement inhibition suggests that the complement system significantly impacts the immune response to this fungus. Mouse models further reinforce this: mice lacking a functioning complement system had a higher mortality rate post-A. fumigatus exposure compared to their complement-capable counterparts [14, 15]. Previous research has also shown a role for CD14 in the release of TNF in response to A. fumigatus stimulation in humans. We do not see a significant effect of CD14 inhibition alone on TNF release. These previous results were, however, done on an isolated myelomonocytic cell line, and the same study showed no role for CD14 in a mouse model [40]. These results stress the importance of holistic models for fully understanding immune and inflammatory responses and the effects of inhibition.

To deepen our understanding of the innate immune responses to A. fumigatus infection, future studies should examine the immune responses to the hyphal form of A. fumigatus. The study of hyphae is especially important as the immune system recognizes conidia and hyphae differently [50], and the conidial form might, therefore, not induce the same cytokine release as hyphae. Furthermore, different strains of A. fumigatus have different virulence and may induce different inflammatory responses [51]. Thus, employing multiple A. fumigatus strains and focusing on the hyphal form will be important in the specific cytokine release patterns during invasive A. fumigatus infections, which will be important to address in future studies.

In conclusion, our findings show that A. fumigatus conidia challenge in whole blood activates complement and induces cell activation, similar to what is observed for E. coli. However, we observed distinct variations in cytokine release levels between the two pathogens. Nevertheless, our results demonstrate that the complement system is a central player in orchestrating cytokine release and support a pivotal role in the early phase of innate immune response against A. fumigatus.

We thank Ms. Bettina Eide Holm, Ms. Sif Kaas Nielsen, and Ms. Victoria Larsen for their excellent technical assistance. We further thank Dr. Laura Perez Alos. Dr. Anne Rosbjerg, and Dr. Verena Harpf for their valuable input and scientific discussions regarding the project.

Human whole blood was obtained from anonymous donors via the Blood Bank at Rigshospitalet in Copenhagen. Informed written consent was not obtained or required in accordance with local and national guidelines. Ethical approval was not required in accordance with Scientific Ethical Committees Act (jf. komitélovens § 14, stk. 3).

The authors have no competing or conflicting interests to declare.

This study was supported by a grant from the EU HORIZON 2020 MSCA ITN project CORVOS 860044.

B. Fageräng, T.E. Mollnes, and P. Garred conceived and designed the research. B. Fageräng, M.P. Götz, L. Cyranka, and C. Lau performed the research and acquired the data. B. Fageräng, M.P. Götz, L. Cyranka, P.H. Nilsson, T.E. Mollnes, and P. Garred analyzed and interpreted the data. B. Fageräng drafted the first version of the manuscript. All authors were involved in the critical revision of the manuscript.

The data that support the findings of this study are not publicly available due to they contain information that could compromise the privacy of research participants but are available from the corresponding author (P.G.) (email address: peter.garred@regionh.dk) upon reasonable request.

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